TCF mutant

ABSTRACT

The present invention relates to TCF mutant having a novel amino acid sequence which is obtained by mutagenesis of one or more amino acid between N-terminus and the first kringle of the amino acid sequence of native TCF and has lowered affinity to heparin and/or elevated biological activity. The present TCF mutant is prepared by gene manipulation of TCF. The TCF mutants of the present invention have proliferative activity and/or growth stimulative activity in hepatocyte and beneficial as a therapeutic agent for various hepatic diseases and an antitumor agent.

FIELD OF THE INVENTION

[0001] The present invention relates to TCF mutants comprising a novelamino acid sequence, more specifically, TCF mutants which are obtainedby mutagenesis of one or more amino acid in the sequence from N-terminusto the first kringle of native TCF and show lowered affinity to heparinand/or elevated biological activity. The TCF mutants of the presentinvention which show proliferative activity and growth stimulativeactivity in hepatocyte are beneficial for treatment of various hepaticdiseases and as an antitumor agent.

BACKGROUND OF THE INVENTION

[0002] Tumor cytotoxic factor (TCF-II) produced in human fibroblastcells is a novel antitumor substance different from any antitumorproteins so far reported. The present inventors have succeeded incloning of cDNA coding for the protein of the present invention,determined the total amino acid sequence thereof and confirmedusefulness thereof (WO90/10651). The molecular weight of TCF was78,000±2,000, or 74,000±2,000 according to the results of SDSelectrophoresis under non-reducing conditions, while the results underreducing conditions indicated A-chain of 52,000±2,000,common band,B-chain of 30,000±2,000 and/or C-chain of 26,000±2,000. TCF is a proteinwhich has a high affinity to heparin or heparin-like substance and showshigh antitumor activity against tumor cells and proliferative activityto normal cells. Further, it was confirmed that it belongs to a widevariety of family of HGF, a growth factor for hepatocyte. Therefore,since TCF is not only an antitumor factor, but also a growth factor forhepatocytes, it is known that it is beneficial for liver regenerationafter hepatectomy.

[0003] Many researches have been carried out from the aspects ofstructure-function relationship of hepatocyte growth factor(HGF) so far.About 20 species of deletion mutants and about 50 species of pointmutants have been reported so far (K. Matsumoto, et. al., Biochem.Biophys. Res. Comm., vol. 181, pp691-699 (1991); G. Hartmann, et. al.Proc. Natl. Acad. Sci. USA, vol. 89, pp11574-11587 -(1992); N. A.Lokker, et. al., EMBO J. vol. 11, pp2503-2510 (1992); M. Okigaki et.al., Biochemistry, vol. 31, pp 9555-9561 (1992); N. A. Lokker, et. al.Protein Engineering, vol. 7, pp895-903 (1994)), however, any mutantwhich clearly shows an elevated biological activity is not obtained atpresent. Half-life of TCF in vivo is known to be extremely short, about2 minutes. Therefore, it is anticipated that a comparatively largeamount of the protein should be administered for treatment of variousdiseases. It is conceivable that the dosage level of TCF administeredwill be reduced by elevation of biological activity thereof or byprolongation of the half-life thereof in vivo. Though it was describedon TCF mutants with prolonged half-life in patent publicationWO94/14845, any TCF mutant with elevated biological activity is notobtained at present, like HGF described above.

[0004] Therefore, the present inventors have investigated to obtain aTCF mutant which shows elevated biological activity or prolongation ofhalf-life in vivo. More specifically, the present inventors have carriedout research to obtain the above-mentioned mutant with elevatedbiological activity or with prolonged half-life in vivo which isdifferent from native TCF with respect to amino acid sequence byaltering the DNA sequence coding for the amino acid sequence of nativeTCF and expressing DNA thereof. Accordingly, an object of the presentinvention is to provide a TCF mutant with elevated biological activityor with prolonged half-life in vivo due to lowered affinity to heparin.

[0005] The present inventors have eagerly investigated on the aboveobject and obtained novel TCF mutants which have amino acid sequencesdifferent from that of TCF mutant found prior to the present inventionand show elevated biological activity and/or lowered affinity toheparin. The present invention provides TCF mutants which show more than10 folds of specific activity (biological activity per unit amount ofprotein) and/or lowered affinity to heparin. These are the first mutantswith extremely elevated biological activity by mutagenizing the aminoacid sequence of native TCF.

SUMMARY OF THE INVENTION

[0006] An object of the present invention is to provide a TCF mutantwith lowered affinity to heparin and/or with elevated biologicalactivity which is obtained by mutagenesis of one or more amino acidresidue(s) in the amino acid sequence from N-terminus to the firstkringle of native TCF.

BRIEF DESCRIPTION OF DRAWINGS

[0007]FIG. 1 shows SDS electrophoresis profiles of purified TCF and TCFmutants of the present invention

[0008]FIG. 2 shows proliferative action of purified TCF and TCF mutantsof the present invention in hepatocyte. The relative activity (%) ofvertical axis is represented as the ratio of proliferative activity ofeach sample based on that of 10 ng/ml TCF as 100%.

[0009]FIG. 3 shows comparison of proliferative action in hepatocytesbetween purified mutant RKRR2AAAA and TCF.

[0010]FIG. 4 shows comparison of proliferative action in hepatocytesbetween purified mutant KIKTKK27AIATAA and TCF.

[0011]FIG. 5 shows comparison of proliferative action in kidneyepithelial cells among purified mutant RKRR2AAAA, mutant KIKTKK27AIATAAand TCF.

[0012]FIG. 6 shows comparison of proliferative action in bone marrowcells among purified mutant RKRR2AAAA, mutant KIKTKK27AIATAA and TCF.

[0013]FIG. 7 shows dose effects of purified TCF, mutant RKRR2AAAA andmutant KIKTKK27AIATAA on the serum level of total protein in rats.

[0014]FIG. 8 shows dose effects of purified TCF, mutant RKRR2AAAA andmutant KIKTKK27AIATAA on the serum level of HDL-cholesterol in rats.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

[0015] By comparing properties of native protein and a mutant obtainedby mutagenesis at some portion of the amino acid sequence of theprotein, function of that portion can be estimated. In the case of aprotein whose structure is not clearly known, it is often used tosubstitute an amino acid, such as Ala, which will not affect the stericstructure for a polar amino acid supposed to be on the surface of aprotein to prevent a structural change of the protein due to themutagenesis. To site-specifically change one amino-acid sequence of aprotein into another, cDNA with site-specific mutations can be preparedby PCR (polymerase chain reaction) method using cDNA coding for nativeTCF as template and synthetic oligonucleotides coding for the otheramino acids. cDNA obtained as described above can be inserted into avector having an appropriate expression promotor (cytomegalovirus (CMV),SRα (Mole. Cell. Biol. vol. 8, No.1, pp466-472 (1988) and JapanesePublished Unexamined Patent Application 277489 (1989) and transfectedinto eukaryotic cells, such as mammalian cells. By culturing thesecells, objective TCF mutants can be prepared from the culture broth.

[0016] Many TCF mutants can be constructed by introducing mutations atdifferent sites or residues. In the present invention, 6 mutants wereprepared. These mutants are specified by enumerating the amino acidsequence before mutagenesis, the number of amino acid at N-terminus ofmutagenized portion and changed amino acid sequence after mutagenesis byone letter code of amino acid. For example, if the whole sequence ofArg-Lys-Arg-Arg at the second position from N-terminus is replaced withAla, the mutant is represented as RKRR2AAAA. For another example, mutantwhose original sequence Lys-Ile-lys-Thr-Lys-lys at 27th position fromN-terminus is replaced with Ala-Ile-Ala-Thr-Ala-Ala is represented asKIKTKK27AIATAA.

[0017] The present invention will be explained in detail by describingexamples. However, these are only exemplified and the scope of theinvention will not be limited by these examples.

EXAMPLE 1

[0018] Site-specific mutation was introduced by the method describedbelow using the 6.3 kb TCF expression plasmid obtained by the methoddescribed in WO92/01053. E. coli comprising this plasmid was depositedas FERM BP-3479.

[0019] I. Preparation of Template Plasmid pcD TCF001

[0020] According to the method below, a mutation was introduced at PstIcleavage site of nucleotide number 34 to change to a nucleotide sequencewhich could not be cleaved. PCR was carried out using 8 ng of plasmidpUC TCF (plasmid in which SalI/SphI fragment of TCF cDNA was insertedinto plasmid pUC18) as a template in the presence of a combination ofmutagenized primer Pst01 (Seq.Id.No.1) and a nonmutagenized primerTCF415 R (Seq.Id.No.2), and in the presence of a combination ofmutagenized primer P002 (Seq.Id.No.3) and a non-mutagenized primerTCFSal-77 (Seq.Id.No.4).

[0021] After the primers were removed from the reaction mixture bymolecular sieving with microcon 100 (Amicon), the products were mixed.And the second PCR was carried out using primer TCFSal-77 and TCF415R.The obtained product was digested by restriction enzymes BstPI and PstI.By using a ligation kit (Takara-shuzo), the fragment was ligated withthe largest BstPI-PstI fragment of pUC TCF BstPI/PstI preparedbeforehand. E.coli DH5α was transformed by using a part of the ligationreaction mixture. Transformed E.coli DH5α was cultured in L brothcontaining 50 μg/ml ampicillin and an objective plasmid was selectedfrom ampicillin resistant colonies. This plasmid was digested byrestriction enzymes SalI and SphI, mixed with new pcDNAI (in whichmulti-cloning site of pcDNAI was mutagenized and there was aHindIII-SalI-BamHI-SphI-NotI cloning site) SalI/SphI large fragmentprepared beforehand and inserted by using a ligation kit. Using thereaction mixture, E.coli MC1061/P3 (Invitrogen) was transformed.Transformed E.coli MC1061/P3 was cultured in L broth containing 50 μg/mlampicillin and 7.5 μg/ml tetracyclin.

[0022] Plasmid DNAs were prepared from obtained ampicillin-tetracyclinresistant colonies and the nucleotide sequence thereof were determinedby a DNA sequencer (Perkin-Elmer). Plasmid pcD TCF001 having anobjective structure was obtained and TCF mutants were prepared by usingthe obtained plasmid.

[0023] II. Construction of an Expression Vector for TCF Mutants andPreparation of Transformed E.coli.

[0024] i. Construction of RKRR2AAAA Expression Vector and Preparation ofTransformed E.coli.

[0025] An expression vector for cDNA coding for RKRR2AAAA wasconstructed by 2 steps of PCR. In the first step, a combination ofmutagenized primer 2RKRRF (Seq.Id.No.5) and non-mutagenized primerTCF977 R (Seq.Id.No.6) and a combination of mutagenized primer 2RKRR R(Seq.Id.No.7) and non-mutagenized primer TCFSal-77 (Seq.Id.No.4) wereused.

[0026] Four nano grams of pcD TCF001 was used as a template in bothreactions. After the reactions, both reaction mixtures were admixed andpurified with microcon 100. One twentieth of the mixture was used astemplate in the second PCR. TCFSal-77 and TCF977 R were used as primers.The reaction mixture was purified with microcon 100 and digested byrestriction enzymes BstPI and EcoRV. By using the ligation kit, thefragment was inserted into the large fragment of an SRα-containing TCFexpression vector cleaved by BstPI and EcoRV beforehand. E.coli DH5α wastransformed with the ligation reaction mixture and an objective clonewas obtained from the obtained ampicillin resistant cells by the samemethod as described before. Plasmid DNA was prepared from the obtainedclone and the DNA sequence thereof was determined by the DNA sequencer(Perkin-Elmer). And this plasmid was cleaved by restriction enzymesEcoRV and BstPI and inserted into the fragment of pUC TCF digested byrestriction enzymes EcoRV and BstPI beforehand, followed bytransformation of E.coli DH5α therewith. E.coli comprising this plasmidwas deposited as pUC TCF2 at National Institute of Bioscience and HumanTechnology on Nov. 10, 1994 and has a deposit number FERM P-14624.

[0027] ii. Construction of KIKTKK27AIATAA Expression Vector andPreparation of Transformed E.coli.

[0028] An expression plasmid for CDNA coding for KIKTKK27AIATAA mutantwas constructed by 2 steps of PCR. In the first PCR, a combination of amutagenized primer 27KIKTKK F (Seq.Id.No.8) and non-mutagenized primerTCF977 R (Seq.Id.No.6) and a combination of mutagenized primer 27KIKTKKR (Seq.Id.No.9) and non-mutagenized primer TCFSal-77 (Seq.Id.No.4) wereused. Four ng of pcD TCF001 was used as a template in both reactions.After the reactions, both reaction mixtures were admixed and purifiedwith microcon 100. One twentieth of the mixture was used as template inthe second PCR. TCFSal-77 and TCF977 R were used as primers.

[0029] The reaction mixture was purified with microcon 100 and digestedby restriction enzymes BstPI and EcoRV. By using a ligation kit, thefragment was inserted into the large fragment of the SR-α-containing TCFexpression vector cleaved by BstPI and EcoRV beforehand. E.coli DH5α wastransformed with the ligation reaction mixture and an objective clonewas obtained from the obtained ampicillin resistant cells by the samemethod as described before. Plasmid DNA was prepared from the obtainedclone and the DNA sequence thereof was determined by DNA sequencer. Andthis plasmid was cleaved by restriction enzymes EcoRV and BstPI andincorporated into a fragment of pUC TCF by digested restriction enzymesEcoRV and BstPI, followed by transformation of E.coli DH5α therewith.E.coli comprising this plasmid was deposited at National Institute ofBioscience and Human-Technology Nov. 10, 1994 and has the deposit numberFERM P-14623.

[0030] iii. Construction of K54A Expression Vector and Preparation ofTransformed E.coli.

[0031] An expression plasmid for cDNA coding for K54A mutant wasconstructed by 2 steps of PCR. In the first PCR, a combination ofmutagenized primer 54K F (Seq.Id.No.10) and non-mutagenized primer TCF977 R (Seq.Id.No.6) and a combination of mutagenized primer 54K R(Seq.Id.No.11) and non-mutagenized primer TCFSal-77 (Seq.Id.No.4) wereused. Four ng of pcD TCF001 was used as a template in both reactions.After the reactions, both reaction mixtures were admixed and purifiedwith microcon 100.

[0032] One twentieth of the mixture was used as template in the secondPCR. TCFSal-77 and TCF 977 R were used as primers. The reaction productwas purified with microcone 100 and digested by restriction enzymesBstPI and EcoRV. By using a ligation kit, the fragment was inserted intothe large fragment of the SRα-containing TCF expression vector cleavedby BstPI and EcoRV beforehand. E.coli DH5α was transformed with theligation reaction mixture and an objective clone was obtained from theobtained ampicillin resistant cells by the same method as describedbefore. Plasmid DNA was prepared from the obtained clone and the DNAsequence thereof was determined by DNA sequencer.

[0033] iv. Construction of RGKD132AGAA Expression Vector and Preparationof Transformed E.coli.

[0034] An expression plasmide for cDNA coding for RGKD132AGAA mutant wasconstructed by 2 steps of PCR. In the first PCR, a combination ofmutagenized primer 132RGKD F (Seq.ID.No.12) and non-mutagenized primerTCF977R (Seq.ID.No.6) and a combination of mutagenized primer 132RGKD R(Seq.ID.No.13) and primer TCF Sal-77 (Seq.ID.No.4) were used. Four ng ofpcD TCF001 was used as a template in both reactions. After the reactionwas through, both reaction mixtures were admixed and purified withmicrocon 100.

[0035] One twentieth of the mixture was used as template in the secondPCR. TCFSal-77 and TCF977 R were used as primers. The reaction productwas purified with microcon 100 and digested by restriction enzymes BstPIand EcoRV. By using a ligation kit, the fragment was inserted into thelarge fragment of the SRa-containing TCF expression vector cleaved byBstPI and EcoRV beforehand. E.coli DH5α was transformed with theligation reaction mixture and an objective clone was obtained from theobtained ampicillin resistant cell lines. Plasmid DNA was prepared fromthe obtained clone in the same way as described before and the basesequence thereof was determined by DNA sequencer.

[0036] v. Construction of R142A Expression Vector and Preparation ofTransformed E.coli

[0037] An expression plasmid for cDNA coding for R142A mutant wasconstructed by 2 steps of PCR. In the first PCR, a combination ofmutagenized primer 142R F (Seq.ID.No.14) and non-mutagenzed primerTCF977 R (Seq.ID.No.6) and a combination of mutagenized primer 142R R(Seq.ID.No.15) and TCFSal-77 (Seq.ID.No.4) were used. Four ng of pcD TCFwas used as template in both reactions. After the reaction was through,both reaction mixtures were admixed and purified with microcon 100.

[0038] Then, one twentieth of the mixture was used as template in thesecond PCR. The reaction mixture was purified with microcon 100 anddigested by restriction enzymes BstPI and EcoRV. By using a ligationkit, the fragment was inserted into the large fragment of theSRα-containing TCF expression vector cleaved by BstPI and EcoRVbeforehand. E.coli DH5α was transformed with the ligation reactionmixture and an objective clone was obtained from the obtained ampicillinresistant cell lines in the same way as described before. The plasmidDNA was prepared from the obtained clone and the DNA sequence thereofwas determined by DNA sequencer.

[0039] vi. Construction of R42A Expression Vector and Preparation ofTransformed E.coli.

[0040] An expression plasmid for cDNA coding for R42A mutant wasconstructed by 2 steps of PCR. In the first PCR, a combination ofmutagenized primer 42R F (Seq.ID.No.16) and non-mutagenized primerTCF977 R (Seq.ID.No.6) and a combination of mutagenized primer 42R R(Seq.ID. No.17) and TCFSal-77 (Seq.ID.No.4) were used. Four ng of pcDTCF001 was used as template in the both reactions.

[0041] After the reaction was through, the both reaction mixtures wereadmixed and purified with microcon 100. One twentieth of the mixture wasused as template in the second PCR. TCFSal-77 and TCF977 R were used asprimers. The reaction mixture was purified with microcon 100 and wasdigested by restriction enzyme BstPI/EcoRV. By using a ligation kit, thefragment was inserted into the large fragment of the SRα-containing TCFexpression vector cleaved by BstPI and EcoRV beforehand. E.coli DH5α wastransformed with the ligation reaction mixture and an objective clonewas obtained from ampicillin resistant cell lines in the same way asdescribed before. The plasmid DNA was prepared from the obtained cloneand the DNA sequence thereof was determined by DNA sequencer.

[0042] III. Preparation and Purification of Expression Plasmids for TCFMutants

[0043] Six species of transformed E.coli comprising the above expressionplasmids were cultured in L broth (400ml) containing 50 μg/ml ampicillinin a shaking incubator at 37° C. overnight, wherein Spectinomycin(Sigma) was added up to a final concentration of 0.3 mg/ml when OD600 ofcultured broth became 1.0. According to the method of Maniatis(Molecular cloning 2nd ed. ppl.21-1.52 (1989), Cold Spring HarborLaboratory), plasmid DNA was isolated by alkaline SDS method and 6species of TCF mutant expression plasmids were purified by cesiumdensity gradient centrifugation method.

[0044] IV. Transfection of TCF Mutant Expression Plasmid into AnimalCell.

[0045] All the mutant expression plasmids were transfected into ChineseHamster Ovary (CHO) Cells. CHO cells (2×10⁶) were suspended in 0.8 mlIMDM medium (Gibco) containing 10% fetal calf serum (FCS) (Gibco), inwhich a solution of 200 μg of expression vector and 10 μg of Blasticidinresistant gene expression plasmid pSV2 bsr (Funakoshi) dissolvedbeforehand in 25 μl of TE (10 mM Tris-HCl (pH8.0)-1 mM EDTA) was furthersuspended. This suspension received electroporation under the conditionsof 330V and 960 μF. After leaving it at room temperature for 10 minutes,it was suspended in 10 ml of IMDM containing 10% FCS medium and culturedat 37° C. in a CO² incubator (5% CO²) for 2 days. Two days after, thesupernatant was collected and the amount of the expressed TCF mutant wasanalyzed by enzyme immunoassay (EIA) (N. Shima, et. al.,Gastro-enterologia Japonica, Vol. 26, No. 4. pp477-482 (1991)) usinganti-TCF monoclonal antibody. It was used as a sample for assayingbiological activity. The cells were harvested from the bottom of flasksby trypsin (Gibco) treatment and the number of viable cells was counted.About 10,000 cells/well were placed in 96-well plates(Nunc) and culturedin 200 μl/well of IMDM medium containing 10% FCS and 5 μg/ml Blastcidinefor 2-3 weeks. 2-3 weeks after, 50 μl aliquot was taken from each welland investigated on the expression of TCF mutant by EIA. Cell clonesexpressing the TCF mutants were grown in 12-well plates and 25 cm²flasks. The cell lines producing TCF mutant were established from CHOcells by the above operation.

[0046] V. Large Scale Cultivation of TCF Mutant Producing Cells

[0047] Mutant producing cells were harvested from 75 cm² flasks bytrypsin treatment when it became confluent and those cells weretransferred into 10 225-cm² flasks containing 100 ml of the medium andcultured for a week. Then the cultured supernatant was collected. Byrepeating this operation once or twice, 1-21 of the cultured broth wasobtained.

[0048] VI. Purification of the TCF mutants

[0049] It was purified by 3 steps as described below.

[0050] i. Heparin-Sepharose CL-6B

[0051] Precipitates were removed from one-two litter of cultured mediumof CHO cells expressing each TCF mutants by centrifugation (2,000 rpm×10min.) of the medium and filtrating the supernatant through a 0.45 μmfilter (German Science). TCF mutant was adsorbed at 4 ml/min. on aheparin-Sepharose CL-6B column (25 mm×120 mm, pharmacia) equilibratedwith 10 mM Tris-HCl (pH 7.5) containing 0.3M NaCl and 0.01% Tween 20.The column was washed with about 500 ml of equilibration buffer and theTCF mutant was eluted by 10 mM Tris-HCl (pH 7.5) containing 2M NaCl and0.01% Tween 20. The eluted solution was fractionated to 4 ml each by afraction collector and the fractions having absorption at 280 nm werecollected.

[0052] ii. Mono S FPLC

[0053] The fraction containing TCF mutant eluted with 2M NaCl wasdialyzed against 10 mM phosphate buffer (pH 7.0) containing 0.15M NaCl,followed by centrifugation (12,000 rpm×90 min.) to remove precipitate.The supernatant containing TCF mutant was passed through on a Mono Scolumn (5 mm×50 mm, Pharmacia) equilibrated with 10 mM phosphate buffer(pH 7.0) containing 0.15 M NaCl and 0.01% Tween 20 at flow rate of 1ml/min. for TCF mutant to be adsorbed thereon. After the column waswashed with about 30 ml of equilibration buffer, TCF mutant was eluted,by changing the flow rate to 0.5 ml/min, with a linear gradient of NaClup to 1.0 M for 60 min.. The eluted solution was fractionated to 5 mleach by a fraction collector and fractions containing TCF mutant wasanalyzed by absorption at 280 nm and EIA and collected.

[0054] iii. Heparin 5-PW FPLC

[0055] To the fraction containing TCF mutant obtained using Mono Scolumn chromatography, 2-fold amount of 10 mM Tris-HCl (pH 7.5)containing 0.01% Tween 20 was added. The solution was passed through aHeparin 5-PW column (5 mm×75 mm TOSOH) 1 ml/min. equilibrated with 10 mMTris-HCl (pH 7.5) containing 0.3M NaCl and 0.01% Tween 20 for TCF mutantto be absorbed thereon. By changing the flow rate to 0.5 ml/min., TCFmutant was eluted with a linear gradient of NaCl up to 2.0 M for 60 min.

[0056] The eluted solution was fractionated to 5 ml each by a fractioncollector. The fraction containing TCF mutant was analyzed by 280 nmabsorption and EIA and collected. Obtained TCF mutant solution wasdialyzed against PBS containing 0.01% of Tween 20 (TPBS) so as to be thefinal purified product. The amount of protein in the final purifiedproduct was determined by Lowry method. The amino acid sequence of TCFmutant RKRR2AAAA and that of mutant KIKTKK27 were represented inSeq.ID.No.18 and in Seq.ID.No.19 respectively.

[0057] VII. SDS-polyacrylamide Gel Electrophoresis of Purified TCFMutant

[0058] Purified TCF mutant (200 ng) was applied on SDS polyacrylamidegel electrophoresis. Schematic representation of electrophoresis of TCFmutant RKRR2AAAA and KIKTKKK27AIATAA, which exhibited 10-fold increasein biological activity as described below, and native TCF was shown inFIG. 1. Both of the results under reducing conditions(in the presence ofβ-mercaptoethanol) and non-reducing conditions (in the absence ofβ-mercaptoethanol) did not show any difference among the three. Inaddition, there was no band but those to be expected from the structureof both TCF mutants.

EXAMPLE 2

[0059] Affinity of TCF and TCF Mutant to Heparin

[0060] I. Heparin-Sepharose CL-6B

[0061] Precipitates were removed from the cultured medium of CHO cellsexpressing each TCF mutant by centrifugation (1,200 g×10 min.) of themedium and by filtrating the supernatant through a 0.22 m filter. Thefiltrated supernatant was charged on a heparin-Sepharose CL-6B column (5mm×5 mm; Pharmacia) equilibrated with TPBS for TCF mutant to be adsorbedthereon. After washing with 3 ml TPBS, TCF mutant was eluted with 1 mlof TPBS containing 0.2-0.3M NaCl, increasing the salt concentrationstepwise. The concentration of TCF mutant in the eluate was analyzed byEIA and the salt concentration of the eluate was defined as affinity ofmutant to heparin.

[0062] II. Heparin 5-PW FPLC

[0063] The cultured broth of CHO cells expressing each TCF mutant (30-60ml) was centrifuged (1,000 g×10 min.), passed through 0.22 μm filter toremove precipitate and applied on a Heparin 5-PW column equilibratedwith 20 mM Tris-HCl buffer solution containing 0.01% Tween 20 at a flowrate of 1.0 ml/min. for TCF mutant to be adsorbed. After washing thecolumn with about 20 ml of equilibration buffer solution and changingthe flow rate to 0.5 ml/min., TCF mutant was eluted with a lineargradient of NaCl up to 1.5 M for 45 minutes. Fractions of 0.5 ml eachwere taken by a fraction collector and the concentration of TCF mutantin each fraction was quantified by EIA and the salt concentration of theelution was defined as affinity of mutant to heparin.

[0064] The results of determination of affinity of these TCF mutant toheparin are shown in table 1. The elution concentration of NaCl fromheparin-Sepharose represents the concentration at which TCF mutant iseluted in the maximum amount. The relative ratio of elutionconcentration is defined as (the elution concentration of NaCl of mutantTCF/that of native TCF). And n.d. means “not determined”. In theexamination with heparin-Sepharose, RKRR2AAAA, KIKTKK27AIATAA, and R42Aexhibited significantly lowered affinity to heparin. Further, in theexamination with heparin 5-PW, it was observed that affinity of themutants to heparin was lowered to around 70% of that of native TCF.TABLE 1 Heparin- Sepharose Heparin 5-PW Relative Elution Elution Ratioof Concentration Concentration Elution of NaCl (M) of NaCl (M)concentration TCF 0.9 1.14 1.00 RKRR2AAAA 0.6 0.78 0.68 KIKTKK27AIATAA0.6 0.82 0.72 R42A 0.7 0.84 0.74 K54A 0.9 1.10 0.96 RGKD132AGAA 0.9 n.d.n.d. R142A 0.9 n.d. n.d.

EXAMPLE 3

[0065] Proliferative Activity of TCF and TCF Mutants on Hepatocyte invitro

[0066] Proliferative activity was investigated by the following method:According to the method of Segren (Method in cell biology, Vol. 13, p29(1976) Academic Press, New York), hepatocyte was isolated from Wisterrats (about 200 g of body weight). The cells (1.0×10⁴/50 μl/well) wereplaced into the wells of 96-well plates (Falcon) and cultured at 37° C.overnight using Williams E medium (Flow Laboratory)containing 10% fetalcalf serum and 10 μM dexamethasone (hereinafter, abbreviated as basemedium). After 24 hours, 10 μl of base medium containing TCF or TCFmutant was added to each well. The plates were incubated at 37° C. foranother 22 hours. After 22 hours, ³H-thymidine (Amersham) was addedthereto so as to be 1 μCi/well, keeping the culture another 2 hours.After then, the cells were washed twice with PBS and harvested bytreatment of 0.5% trypsin followed by collection of the cells in a glassfilter by cell harvester. The radio activity incorporated in each wellwas measured by Matrix 96 (Packard) as the amount of DNA synthesis. Theresults are shown in FIG. 2. Mutant K54A, RGKD132AGAA and R142A had1.4-fold, 2.0-fold and 1.6-fold, respectively, higher biologicalactivity than native TCF at a TCF antigen concentration of 2.5 ng/ml.Further each mutant which had lowered affinity to heparin was determinedby Lowery method. Then the biological activity was compared with regardto the protein concentration exhibiting 50% of maximum proliferativeactivity (ED50) (FIG. 3 and 4).

[0067] As the results, 2 species of protein, that is, RKRR2AAAA andKIKTKK27AIATAA, exhibited more than 10 folds of biological activity perunit amount of protein comparing with that of native TCF.

EXAMPLE 4

[0068] Proliferative Activity of TCF and TCF Mutant in Kidney EpithelialCells

[0069] Proliferative activity in kidney epithelial cell was determinedby the following method:

[0070] OK cells derived from kidney epithelial cell line of AmericanOpossum were placed into each well of 96-well plates so as to be1.0×10⁴/100 μl/well and cultured in DMEM medium containing 10% fetalcalf serum at 37° C. overnight. After then, each well was washed 2-3times with DMEM medium containing no serum. The medium in each well wasreplaced with DMEM medium containing no serum and the culture was keptat 37° C. for another 2 days. Then, the medium in each well was againreplaced with 50 μl of fresh DMEM medium containing no serum and, with50 μl of addition of TCF or TCF mutant diluted with DMED mediumcontaining 0.2% bovine serum albumin, the culture was kept for another24 hours. After 24 hours, H-thymidine was added thereto so as to be 1μCi/well and the culture was kept for another 2 hours. Then, cells werewashed with PBS twice and the cells were harvested by treatment of 0.5%trypsin, followed by collection of the cells in a glassfilter by a cellharvester. The radio activity incorporated in each well was measured byMatrix 96 and determined as the amount of DNA synthesis. The resultswere exhibited in FIG. 5. As the results, it was observed thatbiological activities per unit amount of protein of RKRR2AAAA andKIKTKK27AIATAA in kidney epithelial cell increased more than 2 foldscomparing with that of native TCF.

EXAMPLE 5

[0071] Proliferative Activity of TCF and TCF Mutant in Bone Marrow Cellin vitro Proliferative activity in bone marrow cell was determined bythe following method: NFS-60 cells which are from a mouse bone marrowcell line were placed into each well of 96 well-plate so as to be5.0×10⁴ cells/50 μl/well in RPMI medium containing 10% fetal calf serumand, with addition of 50 μl of TCF or TCF mutant diluted with themedium, cultured at 37° C. for 24 hours. After 24 hours, 10 μl of 5mg/ml MTT (Sigma) was added to each well and the culture was kept foranother 4 hours. Then, 100 μl of 10% SDS/10 mM ammonium chloride wasadded to each well and it was left at room temperature overnight. Afterthat, optical absorbance at 590 nm was measured by Immunoreader NJ-2000(Intermed) as proliferative activity.

[0072] The results were exhibited in FIG. 6. As the results, it wasobserved that biological activities per unit amount of protein ofRKRR2AAAA and KIKTKK27AIATAA in bone marrow cell decrease to ½-{fraction(1/20)} of that of native TCF.

EXAMPLE 6

[0073] In vivo Biological Activity of TCF and TCF Mutants

[0074] In vivo Biological activity was assayed by the following method:TCF or TCF mutant dissolved in PBS containing 0.01% Tween 20 wasintravenously administered through tail (2 ml/kg×2 times/day) in 6 weeksold male Wister rats for 4 days. At the next day to the finaladministration, blood samples were taken from caudal vena cava underether anesthesia and serum thereof were collected by centrifugation(3000 rpm×10 min.) and, in the case of plasma, immediately aftersampling blood, sodium citrate (the final concentration was 0.38%) wasadded thereto followed by centrifugation(3000 rpm×10 min.) to giveplasma. After serum or plasma obtained was preserved in a freezer keptat −30° C., serum level of total protein, albumin, unsaturated ironbinding capacity, total cholesterol, free cholesterol, HDL-cholesteroland phospholipid were analyzed by serum autoanalyzer (Hitachi 7150Autoanalyzer) and plasma level of prothrombin time and fibrinogen wereanalyzed by Auto blood coagulation analyzer KC40 (Amerung). For theseanalysis, the following analyzing kits were used:

[0075] Total protein: Autosera^(TR) TP, Albumin: Autosera^(TR) ALB,Unsaturated iron-binding capacity: Clinimate UIBC, Total cholesterol:Autosera^(TR) CHO-2, Free cholesterol: Autosera^(TR) F-CHO-2,HDL-cholesterol: HDL-C·2 “DAIICHI”, Phospholipid: Autosera^(TR) PL-2,(All the above kits were products of Daiichi-Pure Chemicals Co., Ltd.)Prothrombin time: Orthobrain thromboplastin (Ortho Diagnostic SystemInc.), Fibrinogen: Sun assay Fib (Nitto Boseki Co., Ltd.). As typicalexamples, dose effects thereof on serum level of total protein and onserum level of HDL-cholesterol were exemplified in FIG. 7 and FIG. 8respectively.

[0076] According to the results of statistical analysis of parallel lineassey, with respect to increase of total protein, RKRR2AAAA exhibited2.12 folds of specific activity and KIKIKTKK27AIATAA exhibited 1.37folds of specific activity, comparing to that of native one. Further,with respect to increase HDL-cholesterol, RKRR2AAAA exhibited 1.66 foldsof specific activity and KIKTKK27AIATAA exhibited 1.62 folds of specificactivity, comparing to that of native one.

[0077] Industrial Availabilities

[0078] The present invention is to provide a novel TCF mutant. The TCFmutant of the present invention has proliferative activity and growthstimulative activity in hepatocyte and beneficial for treatment ofvarious hepatic diseases and as an antitumor agent.

1 25 30 NUCLEIC ACID SINGLE LINEAR 1 GCCAGCCTGC TGCTCCAGCA TGTCCTCCTG 3025 NUCLEIC ACID SINGLE LINEAR 2 TGCCACTCTT AGTGATAGAT ACTGT 25 34NUCLEIC ACID SINGLE LINEAR 3 TTTTAAAAGG AAGTCCTTTA TTCCTAGTAC ATCT 34 32NUCLEIC ACID SINGLE LINEAR 4 GGTCGACTAG GCACTGACTC CGAACAGGAT TC 32 32NUCLEIC ACID SINGLE LINEAR 5 CCCTATGCAG AGGACAAGCG GCAGCTGCCA TT 32 23NUCLEIC ACID SINGLE LINEAR 6 ATACCTGAGA ATCCCAACGC TGA 23 33 NUCLEICACID SINGLE LINEAR 7 GAATTCATGA ATTGTATTGG CAGCTGCCGC TTG 33 35 NUCLEICACID SINGLE LINEAR 8 GGCAATAGCA ACCGCAGCTG TGAATACTGC AGACG 35 38NUCLEIC ACID SINGLE LINEAR 9 CAGCTGCGGT TGCTATTGCC AGTGCTGGAT CTATTTTG38 28 NUCLEIC ACID SINGLE LINEAR 10 CCATTCACTT GCGCGGCTTT TGTTTTTG 28 28NUCLEIC ACID SINGLE LINEAR 11 CAAAAACAAA AGCCGCGCAA GTGAATGG 28 36NUCLEIC ACID SINGLE LINEAR 12 GAACACAGCT ATGCGGGTGC AGCCCTACAG GAAAAC 3636 NUCLEIC ACID SINGLE LINEAR 13 GTTTTCCTGT AGGGCTGCAC CCGCATAGCT GTGTTC36 26 NUCLEIC ACID SINGLE LINEAR 14 GAAAACTACT GTGCAAATCC TCGAGG 26 26NUCLEIC ACID SINGLE LINEAR 15 CCTCGAGGAT TTGCACAGTA GTTTTC 26 27 NUCLEICACID SINGLE LINEAR 16 CAATGTGCTA ATGCATGTAC TAGGAAT 27 26 NUCLEIC ACIDSINGLE LINEAR 17 ATTCCTAGTA CATGCATAGC ACATTG 26 723 AMINO ACID SINGLELINEAR 18 Met Trp Val Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln His ValLeu -30 -25 -20 Leu His Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala GluGly Gln -15 -10 -5 -1 1 Ala Ala Ala Ala Asn Thr Ile His Glu Phe Lys LysSer Ala Lys Thr 5 10 15 Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile LysThr Lys Lys Val 20 25 30 Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr ArgAsn Lys Gly Leu 35 40 45 Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys AlaArg Lys Gln Cys 50 55 60 65 Leu Trp Phe Pro Phe Asn Ser Met Ser Ser GlyVal Lys Lys Glu Phe 70 75 80 Gly His Glu Phe Asp Leu Tyr Glu Asn Lys AspTyr Ile Arg Asn Cys 85 90 95 Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly ThrVal Ser Ile Thr Lys 100 105 110 Ser Gly Ile Lys Cys Gln Pro Trp Ser SerMet Ile Pro His Glu His 115 120 125 Ser Tyr Arg Gly Lys Asp Leu Gln GluAsn Tyr Cys Arg Asn Pro Arg 130 135 140 145 Gly Glu Glu Gly Gly Pro TrpCys Phe Thr Ser Asn Pro Glu Val Arg 150 155 160 Tyr Glu Val Cys Asp IlePro Gln Cys Ser Glu Val Glu Cys Met Thr 165 170 175 Cys Asn Gly Glu SerTyr Arg Gly Leu Met Asp His Thr Glu Ser Gly 180 185 190 Lys Ile Cys GlnArg Trp Asp His Gln Thr Pro His Arg His Lys Phe 195 200 205 Leu Pro GluArg Tyr Pro Asp Lys Gly Phe Asp Asp Asn Tyr Cys Arg 210 215 220 225 AsnPro Asp Gly Gln Pro Arg Pro Trp Cys Tyr Thr Leu Asp Pro His 230 235 240Thr Arg Trp Glu Tyr Cys Ala Ile Lys Thr Cys Ala Asp Asn Thr Met 245 250255 Asn Asp Thr Asp Val Pro Leu Glu Thr Thr Glu Cys Ile Gln Gly Gln 260265 270 Gly Glu Gly Tyr Arg Gly Thr Val Asn Thr Ile Trp Asn Gly Ile Pro275 280 285 Cys Gln Arg Trp Asp Ser Gln Tyr Pro His Glu His Asp Met ThrPro 290 295 300 305 Glu Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn Tyr CysArg Asn Pro 310 315 320 Asp Gly Ser Glu Ser Pro Trp Cys Phe Thr Thr AspPro Asn Ile Arg 325 330 335 Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys AspMet Ser His Gly Gln 340 345 350 Asp Cys Tyr Arg Gly Asn Gly Lys Asn TyrMet Gly Asn Leu Ser Gln 355 360 365 Thr Arg Ser Gly Leu Thr Cys Ser MetTrp Asp Lys Asn Met Glu Asp 370 375 380 385 Leu His Arg His Ile Phe TrpGlu Pro Asp Ala Ser Lys Leu Asn Glu 390 395 400 Asn Tyr Cys Arg Asn ProAsp Asp Asp Ala His Gly Pro Trp Cys Tyr 405 410 415 Thr Gly Asn Pro LeuIle Pro Trp Asp Tyr Cys Pro Ile Ser Arg Cys 420 425 430 Glu Gly Asp ThrThr Pro Thr Ile Val Asn Leu Asp His Pro Val Ile 435 440 445 Ser Cys AlaLys Thr Lys Gln Leu Arg Val Val Asn Gly Ile Pro Thr 450 455 460 465 ArgThr Asn Ile Gly Trp Met Val Ser Leu Arg Tyr Arg Asn Lys His 470 475 480Ile Cys Gly Gly Ser Leu Ile Lys Glu Ser Trp Val Leu Thr Ala Arg 485 490495 Gln Cys Phe Pro Ser Arg Asp Leu Lys Asp Tyr Glu Ala Trp Leu Gly 500505 510 Ile His Asp Val His Gly Arg Gly Asp Glu Lys Cys Lys Gln Val Leu515 520 525 Asn Val Ser Gln Leu Val Tyr Gly Pro Glu Gly Ser Asp Leu ValLeu 530 535 540 545 Met Lys Leu Ala Arg Pro Ala Val Leu Asp Asp Phe ValSer Thr Ile 550 555 560 Asp Leu Pro Asn Tyr Gly Cys Thr Ile Pro Glu CysThr Ser Cys Ser 565 570 575 Val Tyr Gly Trp Gly Tyr Thr Gly Leu Ile AsnTyr Asp Gly Leu Leu 580 585 590 Arg Val Ala His Leu Tyr Ile Met Gly AsnGlu Lys Cys Ser Gln His 595 600 605 His Arg Gly Lys Val Thr Leu Asn GluSer Glu Ile Cys Ala Gly Ala 610 615 620 625 Glu Lys Ile Gly Ser Gly ProCys Glu Gly Asp Tyr Gly Gly Pro Leu 630 635 640 Val Cys Glu Gln His LysMet Arg Met Val Leu Gly Val Ile Val Pro 645 650 655 Gly Arg Gly Cys AlaIle Pro Asn Arg Pro Gly Ile Phe Val Arg Val 660 665 670 Ala Tyr Tyr AlaLys Trp Ile His Lys Ile Ile Leu Thr Tyr Lys Val 675 680 685 Pro Gln Ser690 723 AMINO ACID SINGLE LINEAR 19 Met Trp Val Thr Lys Leu Leu Pro AlaLeu Leu Leu Gln His Val Leu -30 -25 -20 Leu His Leu Leu Leu Leu Pro IleAla Ile Pro Tyr Ala Glu Gly Gln -15 -10 -5 -1 1 Arg Lys Arg Arg Asn ThrIle His Glu Phe Lys Lys Ser Ala Lys Thr 5 10 15 Thr Leu Ile Lys Ile AspPro Ala Leu Ala Ile Ala Thr Ala Ala Val 20 25 30 Asn Thr Ala Asp Gln CysAla Asn Arg Cys Thr Arg Asn Lys Gly Leu 35 40 45 Pro Phe Thr Cys Lys AlaPhe Val Phe Asp Lys Ala Arg Lys Gln Cys 50 55 60 65 Leu Trp Phe Pro PheAsn Ser Met Ser Ser Gly Val Lys Lys Glu Phe 70 75 80 Gly His Glu Phe AspLeu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys 85 90 95 Ile Ile Gly Lys GlyArg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys 100 105 110 Ser Gly Ile LysCys Gln Pro Trp Ser Ser Met Ile Pro His Glu His 115 120 125 Ser Tyr ArgGly Lys Asp Leu Gln Glu Asn Tyr Cys Arg Asn Pro Arg 130 135 140 145 GlyGlu Glu Gly Gly Pro Trp Cys Phe Thr Ser Asn Pro Glu Val Arg 150 155 160Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser Glu Val Glu Cys Met Thr 165 170175 Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp His Thr Glu Ser Gly 180185 190 Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro His Arg His Lys Phe195 200 205 Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp Asp Asn Tyr CysArg 210 215 220 225 Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys Tyr Thr LeuAsp Pro His 230 235 240 Thr Arg Trp Glu Tyr Cys Ala Ile Lys Thr Cys AlaAsp Asn Thr Met 245 250 255 Asn Asp Thr Asp Val Pro Leu Glu Thr Thr GluCys Ile Gln Gly Gln 260 265 270 Gly Glu Gly Tyr Arg Gly Thr Val Asn ThrIle Trp Asn Gly Ile Pro 275 280 285 Cys Gln Arg Trp Asp Ser Gln Tyr ProHis Glu His Asp Met Thr Pro 290 295 300 305 Glu Asn Phe Lys Cys Lys AspLeu Arg Glu Asn Tyr Cys Arg Asn Pro 310 315 320 Asp Gly Ser Glu Ser ProTrp Cys Phe Thr Thr Asp Pro Asn Ile Arg 325 330 335 Val Gly Tyr Cys SerGln Ile Pro Asn Cys Asp Met Ser His Gly Gln 340 345 350 Asp Cys Tyr ArgGly Asn Gly Lys Asn Tyr Met Gly Asn Leu Ser Gln 355 360 365 Thr Arg SerGly Leu Thr Cys Ser Met Trp Asp Lys Asn Met Glu Asp 370 375 380 385 LeuHis Arg His Ile Phe Trp Glu Pro Asp Ala Ser Lys Leu Asn Glu 390 395 400Asn Tyr Cys Arg Asn Pro Asp Asp Asp Ala His Gly Pro Trp Cys Tyr 405 410415 Thr Gly Asn Pro Leu Ile Pro Trp Asp Tyr Cys Pro Ile Ser Arg Cys 420425 430 Glu Gly Asp Thr Thr Pro Thr Ile Val Asn Leu Asp His Pro Val Ile435 440 445 Ser Cys Ala Lys Thr Lys Gln Leu Arg Val Val Asn Gly Ile ProThr 450 455 460 465 Arg Thr Asn Ile Gly Trp Met Val Ser Leu Arg Tyr ArgAsn Lys His 470 475 480 Ile Cys Gly Gly Ser Leu Ile Lys Glu Ser Trp ValLeu Thr Ala Arg 485 490 495 Gln Cys Phe Pro Ser Arg Asp Leu Lys Asp TyrGlu Ala Trp Leu Gly 500 505 510 Ile His Asp Val His Gly Arg Gly Asp GluLys Cys Lys Gln Val Leu 515 520 525 Asn Val Ser Gln Leu Val Tyr Gly ProGlu Gly Ser Asp Leu Val Leu 530 535 540 545 Met Lys Leu Ala Arg Pro AlaVal Leu Asp Asp Phe Val Ser Thr Ile 550 555 560 Asp Leu Pro Asn Tyr GlyCys Thr Ile Pro Glu Lys Thr Ser Cys Ser 565 570 575 Val Tyr Gly Trp GlyTyr Thr Gly Leu Ile Asn Tyr Asp Gly Leu Leu 580 585 590 Arg Val Ala HisLeu Tyr Ile Met Gly Asn Glu Lys Cys Ser Gln His 595 600 605 His Arg GlyLys Val Thr Leu Asn Glu Ser Glu Ile Cys Ala Gly Ala 610 615 620 625 GluLys Ile Gly Ser Gly Pro Cys Glu Gly Asp Tyr Gly Gly Pro Leu 630 635 640Val Cys Glu Gln His Lys Met Arg Met Val Leu Gly Val Ile Val Pro 645 650655 Gly Arg Gly Cys Ala Ile Pro Asn Arg Pro Gly Ile Phe Val Arg Val 660665 670 Ala Tyr Tyr Ala Lys Trp Ile His Lys Ile Ile Leu Thr Tyr Lys Val675 680 685 Pro Glu Ser 690 4 AMINO ACID SINGLE LINEAR 20 Arg Lys ArgArg 1 4 AMINO ACID SINGLE LINEAR 21 Ala Ala Ala Ala 1 6 AMINO ACIDSINGLE LINEAR 22 Lys Ile Lys Thr Lys Lys 1 5 6 AMINO ACID SINGLE LINEAR23 Ala Ile Ala Thr Ala Ala 1 5 4 AMINO ACID SINGLE LINEAR 24 Arg Gly LysAsp 1 4 AMINO ACID SINGLE LINEAR 25 Ala Gly Ala Ala 1

1. A TCF mutant which is obtained by mutagenesis of more than one aminoacid residue at the position from N-terminus to the first kringle of theamino acid sequence of native TCF and has lowered affinity to heparinand/or elevated biological activity.
 2. The TCF mutant according toclaim 1, wherein Arg2-Lys-Arg-Arg5 of native TCF is mutagenized toAla-Ala-Ala-Ala.
 3. The TCF mutant according to claim 1, whereinLys27-Ile-Lys-Thr-Lys-Lys32 of native TCF is mutagenized toAla-Ile-Ala-Thr-Ala-Ala.
 4. The TCF mutant according to claim 2 or 3,wherein proliferative activity thereof per unit amount of protein inhepatocyte is more than 10 folds than that of native TCF.
 5. The TCFmutant according to claim 2 or 3, wherein proliferative activity thereofper unit amount of protein in kidney epithelial cell is more than 2folds than that of native TCF.
 6. The TCF mutant according to claim 2 or3, wherein proliferative activity thereof per unit amount of protein inbone marrow cell is ½-{fraction (1/20)} of that of native TCF.
 7. TheTCF mutant according to claim 1, wherein Lys54 of native TCF ismutagenized to Ala.
 8. The TCF mutant according to claim 1, whereinArgl32-Gly-Lys-Aspl35 of native TCF is mutagenized to Ala-Gly-Ala-Ala.9. The TCF mutant according to claim 1, wherein Argl42 of native TCF ismutagenized to Ala.
 10. The TCF mutant according to claim 1, whereinArg42 of native TCF is mutagenized to Ala.